Page 580 - The Toxicology of Fishes

560 The Toxicology of Fishes

Western blotting has been used to detect the Rb protein in a variety of fishes, including medaka,
rainbow trout, English sole (Parophrys vetulus), and even the primitive coelacanth (Latimeria chalumnae)
(Van Beneden and Ostrander, 1994). The complete cloning and sequencing of the Rb gene were first
published for the rainbow trout (Brunelli and Thorgaard, 1999); however, its potential involvement in
chemical carcinogenesis among trout tumor models has not been reported.
The medaka is perhaps the most interesting fish model expressing Rb, as retinoblastomas have been
induced following exposure of newly hatched fry to methylazoxymethanol acetate (MAM) (Ostrander
et al., 1992), making it the only vertebrate model in which retinoblastomas can be induced with regularity.
The gene has now been sequenced and mutations have been reported in MAM-induced eye tumors
(Rotchell et al., 2001a) and methylene chloride induced liver tumors (Rotchell et al., 2001b). Among
feral fishes, the Rb gene has also recently been isolated and characterized from the marine flatfish dab
(Limanda limanda) (du Corbier et al., 2005). Analysis of dab liver adenoma and carcinoma samples
revealed Rb mutations occurring within the conserved domains of the gene.

p53 Gene
The p53 gene contains 10 coding exons and is expressed in all types of human cells examined to date,
albeit mostly at low levels. Moreover, mutations of p53 have been reported in more than 50% of all
human cancers (Giordano et al., 1998). The human protein is 393 amino acids in length, and a nuclear
translocation domain is located near the cyclin-dependent kinase phosphorylation site, which suggests
a cell-cycle-dependent signal for p53 nuclear translocation. Nuclear localization serves an important
function for p53 and is necessary for it to function as a negative regulator of cell proliferation. In some
human cancers, it is the inability of p53 to be transported to the nucleus that is thought to result in
excessive cell proliferation. p53 was the first tumor suppressor gene cloned in fish (Caron de Fromentel
et al., 1992), and it was found to exhibit 90% homology to the predicted amino acid sequence of the
human gene among five domains. Expression of p53 has also been reported in catfish (Ictalurus
punctatus) (Luft et al., 1998), flounder (Platichthys flesus) (Cachot et al., 1998), a goldfish epithelioma
cell line, and a cell line derived from Chinook salmon embryos via Southern blot analysis (Smith et
al., 1988). In an immunohistochemical study, increased expression of p53 was reported in DEN-induced
liver tumors of rivulus (Rivulus ocellatus marmoratus) (Goodwin and Grizzle, 1994a,b). Interestingly,
studies of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced nonhepatic neoplasms from medaka
(Krause et al., 1997) and AFB -induced liver tumors from trout (Bailey et al., 1996) have failed to
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reveal either p53 mutations or altered gene expression (Krause et al., 1997). Results following an
investigation of ultraviolet light inducibility of Oryzias latipes p53 also suggest that the p53 protein
has a different function in lower vertebrates compared with humans (Chen et al., 2001). Recent evidence
in support of this comes from the work of Rau et al. (2006), who measured p53 in a topminnow
hepatocellular carcinoma cell line (PLHC-1) and in both immortalized and primary rainbow trout
hepatocytes. Their study demonstrated a lack of p53 induction by a number of classic mammalian
inducers (chemotherapeutics), consistent with the idea that piscine p53 is not regulated in the same
manner as human p53.

Other Transcription Factors
Other tumor suppressor genes that function as nuclear transcription factors have been reported in
mammalian models (e.g., WT-1, E2F1, PTC, BRCA-1). To date, there has been a single report of WT
involvement in a fish model of cancer—evidenced as increased expression in spontaneous nephroblas-
tomas in Japanese eel (Anguilla japonica) (Nakatsuru et al., 2000). A WT-1 homolog has been found in
zebrafish (Kent et al., 1995), pufferfish (Miles et al., 1998), medaka (GenBank Accession No.
BAC10628), trout (Brunelli et al., 2001), and carp (Ctenopharyngodon idellus) (Wen et al., 2005). A
second WT gene has recently been identified in zebrafish (Bollig et al., 2006). E2F homologs have been
identified in zebrafish (Song et al., 2004) and a PTC homolog in halibut (Paralichthys olivaceus)
(GenBank Accession No. BAC57975) and zebrafish (GenBank Accession No. CAB39726). No fish
BRCA-1 homolog has been reported, although it has been identified in an invertebrate species and is
likely conserved.